In human newborns and other small mammals, cold exposure increases norepinephrine release to stimulate energy expenditure (adaptive non-shivering thermogenesis) mainly in brown adipose tissue (BAT). This process causes a rapid approximately 20-fold increase in type 2 iodothyronine deiodinase (D2), creating tissue-specific thyrotoxicosis in the BAT. A mouse with targeted disruption of the D2 gene (Dio2-/-) is euthyroid, but develops acute hypothermia when exposed to cold. It survives only by the metabolically costly process of shivering. Thus, optimal thermogenesis in brown adipocytes requires the D2-generated increase in intracellular T4 to T3 conversion that saturates the nuclear T3 receptors (TR). The fact that a single TR-saturating dose of T3 restores BAT energy expenditure in Dio2-/- mice in less than 24h indicates that rapidly T3-responsive mechanisms are involved, of which we know surprisingly little. In studies detailed in this submission we will identify these T3-responsive mechanisms, focusing on two major areas of adaptive thermogenesis, adrenergic signal transduction and oxidation of energy substrates. We hypothesize that the Dio2-/- phenotype is due to insufficient mitochondrial uptake and oxidation of energy substrates rather than lower UCP-1-dependent mitochondrial uncoupling. Using a micro-array analysis and real time PCR we have identified in four genes in BAT that were not known to be regulated by T3. The reduced expression of these could explain the thermogenic defect in the Dio2-/- mouse. Two of these genes encode proteins involved in cAMP generation. The other two encode key proteins that regulate the cellular energy homeostasis. Our knowledge about these T3-dependent pathways is crucial because they do not involve mitochondrial uncoupling and therefore could be functionally relevant in tissues other than BAT. Skeletal muscle is the main site of adaptive thermogenesis in large mammals, including adult humans. We hypothesize that skeletal muscle and BAT share the mechanisms by which T3 induces substrate utilization and energy expenditure. These include high metabolic responsiveness to catecholamines and T3, and the recently described cAMP-induced D2 expression in human skeletal muscle cells. Knowledge about this previously unexplored mechanism is important both from the point of view of normal physiology but also to understand the pathophysiology of starvation and diabetes. The loss of D2 under these circumstances due to ubiquitination and proteasomal proteolysis offers an additional potential mechanism to promote energy conservation.